The present invention relates to a process for preparing, and novel chemical complexes comprising the single chain unit of the urokinase type plasminogen activator (xe2x80x9cscuPAxe2x80x9d) and, at least one unit of the soluble urokinase plasminogen activator receptor (xe2x80x9csuPARxe2x80x9d), and use of the resulting scuPA/suPAR cross-linked complex as a fibrinolytic agent in the thromboembolic disorders. The invention is also directed to use of pharmaceutical compositions in prevention and/or treatment of thromoembolic disorders.
Urokinase type plasminogen activator (uPA), widely used in the treatment of thromoembolic diseases, is biologically synthesized as a proehzyme comprising of a single-chain (scuPA). Limited proteolysis of scuPA results in the formation of two chains in the urokinase plasminogen activator (tcuPA), which is considered to be the active form of the enzyme.
An important regulator of uPA is the plasminogen activator inhibitor-1 (PAI-1), which interacts with tcuPA to form an inactive complex. The binding of tcuPA to the receptor uPAR only slightly reduces the susceptibility of tcuPA to the inhibitory effect of PAI-1.
In U.S. Pat. No. 4,996,005 by M. Tsukada et al, it was demonstrated that the combination of single-chain pro-urokinase and plasminogen enhanced the fibrinolytic activity of the single-chain pro-urokinase without causing systemic fibrinolysis. Typical examples of the single chain pro-urokinase used included those with a molecular weight of 50,000 to 55,000 having a single-chain peptide bond structure. And, plasminogen included sources from human or animal serum, plasma, ascites fluid, placenta extract or placenta tissue extract.
U.S. Pat. No. 5,004,609 by S. Hayashi et al., describes a complex of urokinase with a urokinase inhibitor having a molecular weight of 97,5000+3,000 and a method for producing the complex having the ability to dissolve thrombus.
U.S. Pat. No. 5,626,841 by V. Gurewich et al., describes a method of adjunctive therapy to inhibit reocclusion in a patient after thrombolytic treatment by administering to the patient a bolus of pro-urokinase after completion of a thrombolytic treatment and once every 1 to 10 days after the period of risk of occlusion.
Pro-urokinase has also been used in clinical trials for therapeutic thrombolysis in patients with myocardial infarction, pulmonary embolism or deep vein thrombosis. U.S. Pat. No. 5,055,295 by Welzel et al. However, urokinase has a short half-life in blood and lacks noticeable affinity for fibrin. Therefore, its therapeutic use requires bolus-infusion administration in substantial doses which may lead to complications. See, Marder VJ et al. e.g. N. Eng. J. Med. 1988, 318:1512-1520. More effective thrombolysis has been attempted by a generation of plasminogen activator preparations using uPA conjugated with fibrinogen. See Maksimenko Av et al., e.g. Am NY Acad Sci 1990, 613:479-482.
Similarly, in co-pending application, U.S. application Ser. No. 09/325,917 by A. A. Higazi, a combination of a single chain urokinase type plasminogen activator and a soluble urokinase plasminogen activator receptor, for example, the native nature, non-cross-linked scuPA/suPAR complex under physiological conditions was described to be useful as a pharmaceutical composition for prevention and/or treatment of thromboembolic disorders. This composition was described to have improved fibrinolytic activity in the presence of human IgG or at least one human IgG-derived peptide in physiological conditions.
An additional co-pending application (Serial No. unassigned) by A. A. Higazi, describes the two chain urokinase composition (tcuPA) having a fibrinolytic activity when combined with suPAR (tcuPA/suPAR) under physiological conditions.
However, the binding of scuPA to suPAR is reversible and interruption of this binding leads to the loss of activity of the native, non-cross-linking scuPA/suPAR complex. J. Biol. Chem 1995, 270: 17375-17380. Moreover, the role of urokinase (scuPA) and its receptor (suPAR) in fibrinolysis may depend on the vascular location, the physical properties of the clot and its impact on tissue function, and physiological factors contributing to low affinity of suPAR to form dimmers or oligomers that bind scuPA. J.B.C. 2000, 275:24304-24312. The present invention is directed to achieve optimal, cross-linked and/or covalent binding of scuPA to suPAR and to maximize the activity of urokinase in the presence of the chemically prepared single compound, e.g., the cross-linked and/or covalent scuPA/suPAR complex. This is achieved by covalent cross-linked binding of scuPA to suPAR in phosphate buffered saline at 4xc2x0 C. in the presence of sulfosuccinimidyl, instead of relying on the binding to take place under physiological and sometimes not easy to control conditions. The resulting chemical compound has novel properties, for example, it cannot undergo dissociation while at the same time, retains the activity of the corresponding native enzyme-receptor complex, for example, the cross-linked and/or covalent scu/PA/suPAR complex.
The present invention relates to the preparation and use of cross-linked and/or covalently bound single compounds having fibrinolytic activity for example, the scuPA/suPAR complex, comprising of chemically bound single chain urokinase type plasminogen activator (scuPA) and one or more molecules of soluble urokinase plasminogen activator receptors (suPAR).
More specifically, these compounds are useful in the prevention and/or treatment of thromboembolic disorders associated with formation of fibrin clots, for example, myocardial infarctions, celebro-vascular events, pulmonary embolism, peripheral vascular disease, deep vein thrombosis, coronary artery disease, hypercholesterolemia, hypertension, ischemic vascular disease, diabetes, hemoglobinopathies, or myeloproliferative disorders such as thrombocythemia and polycythemia.
The present invention also provides novel processes for the synthesis of cross-linked, covalent complexes of scuPA and suPAR in molar or excess of suPAR concentrations in the presence of sulfosuccinimidyl suborate at specific experimental conditions. The present invention also provides novel processes for the synthesis of cross-linked, covalent complexes of tcuPA/suPAR complex in equimolar or molar excess of suPAR.
Also contemplated by the present invention are methods of dissolving clots in mammals, such methods comprising administering to the mammal a pharmaceutical composition comprising non-cross-linked and/or covalently bound scuPA/suPAR, in an amount therapeutically effective to dissolve the clots, in combination with a physiological buffer.
Also contemplated by the present invention are methods of dissolving clots in mammals, such methods comprising administering to the mammal a pharmaceutical composition comprising non-cross-linked and/or covalently bound scuPA/suPAR, or tcuPA/suPAR in an amount therapeutically effective to dissolve the clots, in combination with a physiological buffer and an effective amount of human IgG.
Pharmaceutical kits for the treatment of thromboembolic disorders in a mammal comprise a sterile container of a pharmaceutical composition comprising cross-linked and/or covalently bound active scuPA/scPAR, or tcuPA/suPAR in an amount therapeutically effective to dissolve clots, in combination with a physiological buffer, and, if desired, mixtures of these, and other activators of plasminogen, comprising streptokinase, rt-PA or alteplase, rt-PA derivatives (such as reteplase, lanoteplase and TNK-rt-PA), platelet glycoprotein IIb-IIIa receptor inhibitors, plasminogen streptokinase complex (APSC) or anistreplase, tcu-PA or urokinase, recombinant scuPA (pro-uPA or prourokinase) and streptokinase and derivatives.
In yet another embodiment, the present invention pertains to processes for stabilizing the cross-linked, covalently bound scuPA/suPAR compound, comprising subjecting the scuPA/suPAR protein, to lyophilization, and then reconstituted in an amount therapeutically effective to prevent and/or treat thromboembolic disorders in a mammal.
Additionally, the present invention is directed to pharmaceutical kits for the treatment of thromboembolic disorders in a mammal, the kits comprising a sterile container of scuPA/suPAR, in lyophilized form, in an amount therapeutically effective to treat thromboembolic disorders, and at least one sterile container of a reconstitution liquid. The foregoing kits may further include, if desired, other fibrinolytic agents such as native non-cross-linked scuPA/suPAR or tcuPA/suPAR complex in an amount therapeutically effective to treat thromboembolic disorders.